Mapping Odor to Action: (Dopaminergic) Timing Is Everything.

Animal brains use the relative timing between sensory cues and behaviorally salient events to form predictive associations about their environment. Handler and colleagues provide new mechanistic insights into how differential signaling downstream of dopamine receptors couples this timing to the dynamic reweighting of synapses that link sensation to action.

Lineages to circuits: the developmental and evolutionary architecture of information channels into the central complex.

The representation and integration of internal and external cues is crucial for any organism to execute appropriate behaviors. In insects, a highly conserved region of the brain, the central complex (CX), functions in the representation of spatial information and behavioral states, as well as the transformation of this information into desired navigational commands. How does this relatively invariant structure enable the incorporation of information from the diversity of anatomical, behavioral, and ecological niches occupied by insects? Here, we examine the input channels to the CX in the context of their development and evolution. Insect brains develop from ~ 100 neuroblasts per hemisphere that divide systematically to form "lineages" of sister neurons, that project to their target neuropils along anatomically characteristic tracts. Overlaying this developmental tract information onto the recently generated Drosophila "hemibrain" connectome and integrating this information with the anatomical and physiological recording of neurons in other species, we observe neuropil and lineage-specific innervation, connectivity, and activity profiles in CX input channels. We posit that the proliferative potential of neuroblasts and the lineage-based architecture of information channels enable the modification of neural networks across existing, novel, and deprecated modalities in a species-specific manner, thus forming the substrate for the evolution and diversification of insect navigational circuits.

Therapeutic applications of trans-splicing.

BACKGROUND: RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein. SOURCES OF DATA: Comprehensive literature search of published research papers and reviews using Pubmed. AREAS OF AGREEMENT: Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction. AREAS OF CONTROVERSY: Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge. GROWING POINTS: Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies. AREAS TIMELY FOR DEVELOPING RESEARCH: Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.

Asymmetric neurotransmitter release enables rapid odour lateralization in Drosophila.

In Drosophila, most individual olfactory receptor neurons (ORNs) project bilaterally to both sides of the brain. Having bilateral rather than unilateral projections may represent a useful redundancy. However, bilateral ORN projections to the brain should also compromise the ability to lateralize odours. Nevertheless, walking or flying Drosophila reportedly turn towards the antenna that is more strongly stimulated by odour. Here we show that each ORN spike releases approximately 40% more neurotransmitter from the axon branch ipsilateral to the soma than from the contralateral branch. As a result, when an odour activates the antennae asymmetrically, ipsilateral central neurons begin to spike a few milliseconds before contralateral neurons, and at a 30 to 50% higher rate than contralateral neurons. We show that a walking fly can detect a 5% asymmetry in total ORN input to its left and right antennal lobes, and can turn towards the odour in less time than it requires the fly to complete a stride. These results demonstrate that neurotransmitter release properties can be tuned independently at output synapses formed by a single axon onto two target cells with identical functions and morphologies. Our data also show that small differences in spike timing and spike rate can produce reliable differences in olfactory behaviour.

Determination of the True Inclination Angle of the Main Bronchi Relative to the Median Sagittal Plane for Placement of a Left-Sided Double-Lumen Tube.

OBJECTIVES: This study aimed to determine the true inclination angle of the main bronchi relative to the median sagittal plane, using CT imaging to help increase accuracy of double-lumen tube (DLT) placement. DESIGN: In this retrospective study, 2 investigators independently measured normal chest CT scans from 50 male and 50 female patients. To determine the true AP axis, a mid-sagittal plane reference line (MSPRL) was drawn, intersecting the midsternum and the vertebral spinous process at the level of mid-carina. Lines were drawn through the center of each main bronchus to determine the inclination angle with regard to the MSPRL. SETTING: Research was conducted at a single institution, the Los Angeles County and University of Southern California Medical Center. PARTICIPANTS: Normal chest CT images from 50 women and 50 men. MAIN RESULTS: The mean true inclination angle between the main bronchi and trachea in the mid-sagittal plane was 108.4° on the left compared with 96.2° on the right (p<0.0001). INTERVENTIONS: No specific interventions were done because this was a retrospective study and CT scan analysis. CONCLUSION: The data suggested that the trachea does not merely branch in the horizontal plane but branches posteriorly as well, with a true mean anatomic angle between the left main bronchus and trachea of 108.4°. This finding concurred with the authors' suggestion that the DLT be rotated to 110° counterclockwise instead of the routine practice of 90°. The authors suggest clinicians rotate the DLT an additional 20° counterclockwise and direct the top of the DLT to the 11 o'clock position.

MLML: consistent simultaneous estimates of DNA methylation and hydroxymethylation.

MOTIVATION: The two major epigenetic modifications of cytosines, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), coexist with each other in a range of mammalian cell populations. Increasing evidence points to important roles of 5-hmC in demethylation of 5-mC and epigenomic regulation in development. Recently developed experimental methods allow direct single-base profiling of either 5-hmC or 5-mC. Meaningful analyses seem to require combining these experiments with bisulfite sequencing, but doing so naively produces inconsistent estimates of 5-mC or 5-hmC levels. RESULTS: We present a method to jointly model read counts from bisulfite sequencing, oxidative bisulfite sequencing and Tet-Assisted Bisulfite sequencing, providing simultaneous estimates of 5-hmC and 5-mC levels that are consistent across experiment types. AVAILABILITY: http://smithlab.usc.edu/software/mlml

Antiviral defense in plant stem cells.

Undifferentiated plant and animal stem cells are essential for cell, tissue, and organ differentiation, development, and growth. They possess unusual antiviral immunity which differs from that in specialized cells. By comparison to animal stem cells, we discuss how plant stem cells defend against viral invasion and beyond.

Chronic exposure to odors at naturally occurring concentrations triggers limited plasticity in early stages of Drosophila olfactory processing.

In insects and mammals, olfactory experience in early life alters olfactory behavior and function in later life. In the vinegar fly Drosophila, flies chronically exposed to a high concentration of a monomolecular odor exhibit reduced behavioral aversion to the familiar odor when it is reencountered. This change in olfactory behavior has been attributed to selective decreases in the sensitivity of second-order olfactory projection neurons (PNs) in the antennal lobe that respond to the overrepresented odor. However, since odorant compounds do not occur at similarly high concentrations in natural sources, the role of odor experience-dependent plasticity in natural environments is unclear. Here, we investigated olfactory plasticity in the antennal lobe of flies chronically exposed to odors at concentrations that are typically encountered in natural odor sources. These stimuli were chosen to each strongly and selectively excite a single class of primary olfactory receptor neuron (ORN), thus facilitating a rigorous assessment of the selectivity of olfactory plasticity for PNs directly excited by overrepresented stimuli. Unexpectedly, we found that chronic exposure to three such odors did not result in decreased PN sensitivity but rather mildly increased responses to weak stimuli in most PN types. Odor-evoked PN activity in response to stronger stimuli was mostly unaffected by odor experience. When present, plasticity was observed broadly in multiple PN types and thus was not selective for PNs receiving direct input from the chronically active ORNs. We further investigated the DL5 olfactory coding channel and found that chronic odor-mediated excitation of its input ORNs did not affect PN intrinsic properties, local inhibitory innervation, ORN responses or ORN-PN synaptic strength; however, broad-acting lateral excitation evoked by some odors was increased. These results show that PN odor coding is only mildly affected by strong persistent activation of a single olfactory input, highlighting the stability of early stages of insect olfactory processing to significant perturbations in the sensory environment.

Restructuring of olfactory representations in the fly brain around odor relationships in natural sources.

A core challenge of olfactory neuroscience is to understand how neural representations of odor are generated and progressively transformed across different layers of the olfactory circuit into formats that support perception and behavior. The encoding of odor by odorant receptors in the input layer of the olfactory system reflects, at least in part, the chemical relationships between odor compounds. Neural representations of odor in higher order associative olfactory areas, generated by random feedforward networks, are expected to largely preserve these input odor relationships1-3. We evaluated these ideas by examining how odors are represented at different stages of processing in the olfactory circuit of the vinegar fly D. melanogaster. We found that representations of odor in the mushroom body (MB), a third-order associative olfactory area in the fly brain, are indeed structured and invariant across flies. However, the structure of MB representational space diverged significantly from what is expected in a randomly connected network. In addition, odor relationships encoded in the MB were better correlated with a metric of the similarity of their distribution across natural sources compared to their similarity with respect to chemical features, and the converse was true for odor relationships encoded in primary olfactory receptor neurons (ORNs). Comparison of odor coding at primary, secondary, and tertiary layers of the circuit revealed that odors were significantly regrouped with respect to their representational similarity across successive stages of olfactory processing, with the largest changes occurring in the MB. The non-linear reorganization of odor relationships in the MB indicates that unappreciated structure exists in the fly olfactory circuit, and this structure may facilitate the generalization of odors with respect to their co-occurence in natural sources.

High concordance between HercepTest immunohistochemistry and ERBB2 fluorescence in situ hybridization before and after implementation of American Society of Clinical Oncology/College of American Pathology 2007 guidelines.

Human epidermal growth factor receptor 2 (HER2, ERBB2) is an important critical predictive marker in patients with invasive breast cancer. It is thus imperative to ensure accuracy and precision in HER2 and ERBB2 testing. In 2007, the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) proposed new guidelines for immunohistochemistry and fluorescence in-situ hybridization scoring in an effort to improve accuracy and utility of these companion diagnostic tests. The goal of the 2007 guidelines was to improve concordance rates between the diagnostic tests and decrease the number of inconclusive cases. This study examines the impact in concordance rates and number of inconclusive cases based on the recent change in guidelines in a large study cohort. HER2 immunohistochemistry and ERBB2 fluorescence in-situ hybridization were performed on all specimens from our facility from years 2003 through 2010 (n=1437). Cases from 2003-2007 (n=1016) were scored using Food and Drug Administration guidelines, with immunohistochemical 3+ cases staining >10% of tumor cells and fluorescence in-situ hybridization amplification cutoff value of 2.0. The 2007 guidelines were implemented and scored accordingly for cases from 2008-2010 (n=421), with immunohistochemical 3+ cases staining >30% of tumor cells and fluorescence in-situ hybridization amplification cutoff value of 2.2. We compared concordance rates before and after 2007 guidelines. For the 2003-2007 study population, the concordance rate between the assays was 97.6% with a corresponding kappa coefficient (k) of 0.90. For the 2008-2010 study population, concordance rate was 97.6% with a corresponding k of 0.89. There was no significant difference in number of inconclusive rates before and after 2007 guidelines. In our study, implementation of the new ASCO/CAP 2007 HER2 guidelines did not show a significant difference in concordance rates and did not decrease the number of inconclusive cases.

Developmental axon pruning mediated by BDNF-p75NTR-dependent axon degeneration.

The mechanisms that regulate the pruning of mammalian axons are just now being elucidated. Here, we describe a mechanism by which, during developmental sympathetic axon competition, winning axons secrete brain-derived neurotrophic factor (BDNF) in an activity-dependent fashion, which binds to the p75 neurotrophin receptor (p75NTR) on losing axons to cause their degeneration and, ultimately, axon pruning. Specifically, we found that pruning of rat and mouse sympathetic axons that project to the eye requires both activity-dependent BDNF and p75NTR. p75NTR and BDNF are also essential for activity-dependent axon pruning in culture, where they mediate pruning by directly causing axon degeneration. p75NTR, which is enriched in losing axons, causes axonal degeneration by suppressing TrkA-mediated signaling that is essential for axonal maintenance. These data provide a mechanism that explains how active axons can eliminate less-active, competing axons during developmental pruning by directly promoting p75NTR-mediated axonal degeneration.

Olfactory coding.

Modern humans live in real and digital environments dominated by sight and sound, but the vast majority of organisms on the planet rely on information received through air- or water-borne molecules to find food, avoid danger, and reproduce. Olfaction is at once both the primitive sensory modality and one of the hardest to understand, in large part due to the complexity of olfactory stimulus space. Whereas light and sound are easily ordered along natural physical axes that are reflected in their respective sensory codes, the organizational axes of odor space are not obvious. The search for systematic relationships between physicochemical characteristics of monomolecular odorants (carbon chain length, bond numbers, functional groups, etc.) and human perception of odorants suggests that olfactory perceptual space is a relatively low-dimensional structure. Odor descriptors provided by human observers are often significantly correlated. For instance, odors perceived as 'woody' are also likely to be described as 'warm', and many studies converge on hedonic valence or 'pleasantness' as being one of the most important dimensions of how people perceive odors. The identification of additional perceptual 'primaries' around which olfaction is organized is an active area of investigation, and a useful account of olfactory coding must explain this transformation of odor stimuli from the high dimensional chemical space to a lower dimensional perceptual space.

Parallel encoding of CO2 in attractive and aversive glomeruli by selective lateral signaling between olfactory afferents.

We describe a novel form of selective crosstalk between specific classes of primary olfactory receptor neurons (ORNs) in the Drosophila antennal lobe. Neurotransmitter release from ORNs is driven by two distinct sources of excitation: direct activity derived from the odorant receptor and stimulus-selective lateral signals originating from stereotypic subsets of other ORNs. Consequently, the level of presynaptic neurotransmitter release from an ORN can be significantly dissociated from its firing rate. Stimulus-selective lateral signaling results in the distributed representation of CO2-a behaviorally important environmental cue that directly excites a single ORN class-in multiple olfactory glomeruli, each with distinct response dynamics. CO2-sensitive glomeruli coupled to behavioral attraction respond preferentially to fast changes in CO2 concentration, whereas those coupled to behavioral aversion more closely follow absolute levels of CO2. Behavioral responses to CO2 also depend on the temporal structure of the stimulus: flies walk upwind to fluctuating, but not sustained, pulses of CO2. Stimulus-selective lateral signaling generalizes to additional odors and glomeruli, revealing a subnetwork of lateral interactions between ORNs that reshapes the spatial and temporal structure of odor representations in a stimulus-specific manner.

Polarized signaling endosomes coordinate BDNF-induced chemotaxis of cerebellar precursors.

During development, neural precursors migrate in response to positional cues such as growth factor gradients. However, the mechanisms that enable precursors to sense and respond to such gradients are poorly understood. Here we show that cerebellar granule cell precursors (GCPs) migrate along a gradient of brain-derived neurotrophic factor (BDNF), and we demonstrate that vesicle trafficking is critical for this chemotactic process. Activation of TrkB, the BDNF receptor, stimulates GCPs to secrete BDNF, thereby amplifying the ambient gradient. The BDNF gradient stimulates endocytosis of TrkB and associated signaling molecules, causing asymmetric accumulation of signaling endosomes at the subcellular location where BDNF concentration is maximal. Thus, regulated BDNF exocytosis and TrkB endocytosis enable precursors to polarize and migrate in a directed fashion along a shallow BDNF gradient.

Perineural dexmedetomidine added to ropivacaine for sciatic nerve block in rats prolongs the duration of analgesia by blocking the hyperpolarization-activated cation current.

BACKGROUND: The current study was designed to test the hypothesis that the increased duration of analgesia caused by adding dexmedetomidine to local anesthetic results from blockade of the hyperpolarization-activated cation (I(h)) current. METHODS: In this randomized, blinded, controlled study, the analgesic effects of peripheral nerve blocks using 0.5% ropivacaine alone or 0.5% ropivacaine plus dexmedetomidine (34 µM or 6 µg/kg) were assessed with or without the pretreatment of α(1)- and α(2)-adrenoceptor antagonists (prazosin and idazoxan, respectively) and antagonists and agonists of the I(h) current (ZD 7288 and forskolin, respectively). Sciatic nerve blocks were performed, and analgesia was measured by paw withdrawal latency to a thermal stimulus every 30 min for 300 min postblock. RESULTS: The analgesic effect of dexmedetomidine added to ropivacaine was not reversed by either prazosin or idazoxan. There were no additive or attenuated effects from the pretreatment with ZD 7288 (I(h) current blocker) compared with dexmedetomidine added to ropivacaine. When forskolin was administered as a pretreatment to ropivacaine plus dexmedetomidine, there were statistically significant reductions in duration of analgesia at time points 90-180 min (P < 0.0001 for each individual comparison). The duration of blockade for the forskolin (768 µM) followed by ropivacaine plus dexmedetomidine group mirrored the pattern of the ropivacaine alone group, thereby implying a reversal effect. CONCLUSION: Dexmedetomidine added to ropivacaine caused approximately a 75% increase in the duration of analgesia, which was reversed by pretreatment with an I(h) current enhancer. The analgesic effect of dexmedetomidine was not reversed by an α(2)-adrenoceptor antagonist.

Nursing interventions to promote dignified dying in South Korea.

PURPOSE: This descriptive study aimed to assess the appropriateness of the International Classification for Nursing Practice (ICNP) catalogue Palliative Care for Dignified Dying for palliative care nursing interventions in South Korea. METHODS: The study surveyed 213 South Korean nurses who might regularly care for dying patients. Nurses were recruited to complete a survey that included interventions from the ICNP catalogue listed with Likert response sets. FINDINGS: All of the interventions were scored as being at least 'slightly important' on average. The following three nursing interventions were ranked as most important when caring for dying patients: establish trust, establish rapport, and administer pain medication. CONCLUSIONS: The study provides new insights into the palliative care provided in South Korea by documenting nurses' views of what are the most important palliative care nursing interventions. It also suggests that the palliative care interventions listed in the ICNP catalogue Palliative Care for Dignified Dying are in congruence with the interventions that nurses in South Korea use.

Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation.

Mutations or duplications in MECP2 cause Rett and Rett-like syndromes, neurodevelopmental disorders characterized by mental retardation, motor dysfunction, and autistic behaviors. MeCP2 is expressed in many mammalian tissues and functions as a global repressor of transcription; however, the molecular mechanisms by which MeCP2 dysfunction leads to the neural-specific phenotypes of RTT remain poorly understood. Here, we show that neuronal activity and subsequent calcium influx trigger the de novo phosphorylation of MeCP2 at serine 421 (S421) by a CaMKII-dependent mechanism. MeCP2 S421 phosphorylation is induced selectively in the brain in response to physiological stimuli. Significantly, we find that S421 phosphorylation controls the ability of MeCP2 to regulate dendritic patterning, spine morphogenesis, and the activity-dependent induction of Bdnf transcription. These findings suggest that, by triggering MeCP2 phosphorylation, neuronal activity regulates a program of gene expression that mediates nervous system maturation and that disruption of this process in individuals with mutations in MeCP2 may underlie the neural-specific pathology of RTT.

Brain-derived neurotrophic factor expression and respiratory function improve after ampakine treatment in a mouse model of Rett syndrome.

Rett syndrome (RTT) is caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is thought to act as a transcriptional repressor of brain-derived neurotrophic factor (BDNF), Mecp2 null mice, which develop an RTT-like phenotype, exhibit progressive deficits in BDNF expression. These deficits are particularly significant in the brainstem and nodose cranial sensory ganglia (NGs), structures critical for cardiorespiratory homeostasis, and may be linked to the severe respiratory abnormalities characteristic of RTT. Therefore, the present study used Mecp2 null mice to further define the role of MeCP2 in regulation of BDNF expression and neural function, focusing on NG neurons and respiratory control. We find that mutant neurons express significantly lower levels of BDNF than wild-type cells in vitro, as in vivo, under both depolarizing and nondepolarizing conditions. However, BDNF levels in mutant NG cells can be increased by chronic depolarization in vitro or by treatment of Mecp2 null mice with CX546, an ampakine drug that facilitates activation of glutamatergic AMPA receptors. Ampakine-treated Mecp2 null mice also exhibit marked functional improvement, characterized by restoration of normal breathing frequency and minute volume. These data demonstrate that BDNF expression remains plastic in Mecp2 null mice and raise the possibility that ampakine compounds could be of therapeutic value in the treatment of RTT.

Transcriptional control of cognitive development.

Cognitive development is determined by both genetics and environment. One point of convergence of these two influences is the neural activity-dependent regulation of programs of gene expression that specify neuronal fate and function. Human genetic studies have linked several transcriptional regulators to neurodevelopmental disorders including mental retardation and autism spectrum disorders. Recent reports on two such factors, CREB-binding protein and methyl-CpG-binding protein 2, have begun to reveal how epigenetics and neuronal activity act to modulate the program of gene expression required for synaptic development and function.

Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT).

Understanding the computations that take place in brain circuits requires identifying how neurons in those circuits are connected to one another. We describe a technique called TRACT (TRAnsneuronal Control of Transcription) based on ligand-induced intramembrane proteolysis to reveal monosynaptic connections arising from genetically labeled neurons of interest. In this strategy, neurons expressing an artificial ligand ('donor' neurons) bind to and activate a genetically-engineered artificial receptor on their synaptic partners ('receiver' neurons). Upon ligand-receptor binding at synapses the receptor is cleaved in its transmembrane domain and releases a protein fragment that activates transcription in the synaptic partners. Using TRACT in Drosophila we have confirmed the connectivity between olfactory receptor neurons and their postsynaptic targets, and have discovered potential new connections between neurons in the circadian circuit. Our results demonstrate that the TRACT method can be used to investigate the connectivity of neuronal circuits in the brain.